The cytoskeleton, a cytoplasmic system of protein fibers, mediates cell shape, structure, and movement. The cytoskeleton supports the cell membrane and forms tracks along which organelles and other elements move in the cytosol. The cytoskeleton is a dynamic structure that allows cells to adopt various shapes and to carry out directed movements. Major cytoskeletal fibers are the microfilaments, the microtubules, and the intermediate filaments. Motor proteins including myosin, dynein, and kinesin, drive movement of or along the fibers. Accessory or associated proteins modify the structure or stability of the fibers while cytoskeletal membrane anchors connect the fibers to the cell membrane.
Microtubules, cytoskeletal fibers with a diameter of 24 nm, have multiple roles in the cell. Bundles of microtubules form cilia and flagella, which are whip-like extensions of the cell membrane that are necessary for sweeping materials across an epithelium and for swimming of sperm, respectively. Marginal bands of microtubules in red blood cells and platelets are important for these cells' pliability. Organelles, membrane vesicles, and proteins are transported throughout the cell by motor proteins running along tracks of microtubules. For example, microtubules run through nerve cell axons, allowing bi-directional transport of materials and membrane vesicles between the cell body and the nerve terminal. Failure to supply the nerve terminal with these vesicles blocks the transmission of neural signals. During cell migration, differentiation, and the cell cycle, the microtubule cytoskeleton must rapidly reorganize through assembly and disassembly. Microtubules, in the form of the spindle, are also critical to chromosomal movement during cell division. Both stable and short-lived populations of microtubules exist in the cell.
Microtubules are a polymer of GTP-binding tubulin protein subunits. Each subunit is a heterodimer of α- and β-tubulin, multiple isoforms of which exist. Tubulin binds two molecules of GTP at two different sites, and all three tubulins share an invariant glycine rich region thought to be involved in controlling access to one of the nucleotide binding sites (PROSITE PDOC00199). The tubulins share a motif with FtsZ, a polymer-forming GTPase necessary for bacterial cell division (PROSITE PDOC00873). Alpha-tubulin undergoes a number of post-translational modifications, including acetylation, polyglutamylation, truncation of two amino acids (forming Δ2 tubulin), and tyrosination. In some cases, these modifications can affect microtubule stability. In addition, β-tubulin auto-regulates the stability of its own mRNA. Free β-tubulin binds a conserved motif at the N-terminus of a nascent beta-tubulin chain. This activates an RNAse which degrades the polysome-bound mRNA (PROSITE PDOC00200). The hydrolysis of GTP is linked to the addition of tubulin subunits at the end of a microtubule. The subunits interact head to tail to form protofilaments; the protofilaments interact side to side to form a microtubule. A microtubule is polarized, one end ringed with α-tubulin and the other with β-tubulin, and the two ends differ in their rates of assembly. Microtubules constantly “treadmill,” exchanging their subunits with pools of free tubulin in the cytoplasm. This flow of subunits from one end of the microtubule to the other is created by differences in the critical subunit concentrations at opposite ends of the microtubule and is responsible for the dynamics of microtubule arrays (Margolis, R. L. and L. Wilson (1998) Bioessays 20:830-836). Each microtubule is generally composed of 13 protofilaments although 11 or 15 protofilament-microtubules are sometimes found. Cilia and flagella contain doublet microtubules. Microtubules grow from specialized structures known as centrosomes or microtubule-organizing centers (MTOCs). MTOCs may contain one or two centrioles, which are pinwheel arrays of triplet microtubules. The basal body, the organizing center located at the base of a cilium or flagellum, contains one centriole. γ-tubulin present in the MTOC is important for nucleating the polymerization of α- and β-tubulin heterodimers but does not polymerize into microtubules.
Altered microtubule structure are correlated with numerous neuropathologies, including Alzheimer's disease (Terry, R. D. (1998) J. Neural Transm. Suppl. 53:141-145). An extraskeletal myxoid chondrosarcoma tumor from human contained parallel arrays of microtubules within the rough endoplasmic reticulum (Suzuki, T. et al. (1988) J. Pathol. 156:51-57). Microtubular aggregates were also found in hepatocytes from chimpanzees infected with hepatitis C virus. Altered mictrotubule structures are proposed to have a role in lens opacification (Clark, J. I. et al. (1999) Eye 13:417-424). Microtubules are also a proven target for anti-tumor drugs (Davis, A. et al. (1999) Neoplasia 1:498-507; for a review, see Jordan, A. et al. (1998) Med. Res. Rev. 18:259-296).
Microtubule-associated proteins (MAPs) have roles in the assembly and stabilization of microtubules, one of the major cytoskeletal fibers. A major family of MAPs, assembly MAPs, can be identified in neurons as well as non-neuronal cells. Assembly MAPs are responsible for cross-linking microtubules in the cytosol. These MAPs are organized into two domains: a basic microtubule-binding domain and an acidic projection domain. The projection domain is the binding site for membranes, intermediate filaments, or other microtubules. Based on sequence analysis, assembly MAPs can be further grouped into two types: Type I and Type II.
Type I MAPs, which include MAP1A and MAP1B, are large, filamentous molecules that co-purify with microtubules and are abundantly expressed in brain and testis. They contain several repeats of a positively-charged amino acid sequence motif that binds and neutralizes negatively charged tubulin, leading to stabilization of microtubules. MAP1A and MAP1B are each derived from a single precursor polypeptide that is subsequently proteolytically processed to generate one heavy chain and one light chain.
Another light chain, LC3, is a 16.4 kDa molecule that binds MAP1A, MAP1B, and microtubules. It is suggested that LC3 is synthesized from a source other than the MAP1A or MAP1B transcripts, and the expression of LC3 may be important in regulating the microtubule binding activity of MAP1A and MAP1B during cell proliferation (Mann, S. S. et al. (1994) J. Biol. Chem. 269:11492-11497).
Type II MAPs, which include MAP2a, MAP2b, MAP2c, MAP4, and Tau, are characterized by three to four copies of an 18-residue sequence in the microtubule-binding domain. MAP2a, MAP2b, and MAP2c are found only in dendrites, MAP4 is found in non-neuronal cells, and Tau is found in axons and dendrites of nerve cells. Alternative splicing of the Tau mRNA leads to the existence of multiple forms of Tau protein. Tau phosphorylation is altered in neurodegenerative disorders such as Alzheimer's disease, Pick's disease, progressive supranuclear palsy, corticobasal degeneration, and familial frontotemporal dementia and Parkinsonism linked to chromosome 17. The altered Tau phosphorylation leads to a collapse of the microtubule network and the formation of intraneuronal Tau aggregates (Spillantini, M. G. and Goedert, M. (1998) Trends Neurosci. 21:428-433).
Microtubular aggregates are associated with several disorders. An extraskeletal myxoid chondrosarcoma tumor from human contained parallel arrays of microtubules within the rough endoplasmic reticulum (Suzuki, T. et al. (1988) J. Pathol. 156:51-57). Microtubular aggregates were also found in hepatocytes from chimpanzees infected with hepatitis C virus. Monoclonal antibodies prepared to these aggregates detect a protein called p44 (or microtubular aggregates protein) (Maeda, T. et al. (1989) J. Gen. Virol. 70:1401-1407). A human homolog of p44 is inducible by interferon-α and interferon-β, but not by interferon-γ. p44 protein may be a mediator in the antiviral action of interferon (Kitamura, A. et al. (1994) Eur. J. Biochem. 224:877-883).
The discovery of new microtubule-associated proteins and tubulins, and the polynucleotides encoding them, satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, autoimmune/inflammatory, vesicle trafficking, neurological, cell motility, reproductive, and muscle disorders, and in the assessment of the effects of exogenous compounds on the expression of nucleic acid and amino acid sequences of microtubule-associated proteins and tubulins.